基于高通量转录组测序的牦牛和犏牛附睾尾部差异表达基因分析

Differentially expressed genes between epididymal cauda of yak and cattleyak based on high-throughput transcriptome sequencing

【目的】探明牦牛和犏牛的附睾尾部差异表达基因(DEGs),筛选出与精子成熟和贮存密切相关的功能基因,为揭示犏牛精子发生及其成熟过程的分子机制打下理论基础。【方法】以牦牛和犏牛的附睾尾部为研究对象,通过Illumina 127-HiSeq 2000平台完成高通量转录组测序,经过滤、质量控制及拼接组装后,依据FDR<0.05且|log_(2)Fold Change|>1的筛选标准,通过EBSeq筛选出DEGs,然后进行GO功能注释分析和KEGG信号通路富集分析,并以实时荧光定量PCR验证高通量转录组测序数据的准确性。【结果】在牦牛和犏牛的附睾尾部共筛选获得76个DEGs,其中43个DEGs上调、33个DEGs下调;76个DEGs在COG、GO、KEGG、KOG、Nr、Pfam、Swiss-Prot和eggNOG等数据库中均有注释信息,尤其在COG和Pfam数据库中的注释率最高(达88.16%)。GO功能注释分析结果显示,DEGs被注释到生物过程(Biological process)、细胞组分(Cellular component)和分子功能(Molecular function)三大功能类别上;KEGG信号通路富集分析发现76个DEGs主要显著富集在3条信号通路上,分别是胆汁分泌通路(ko04976:Bile secretion)、ABC转运器(ko02010:ABC transporters)和cAMP信号通路(ko04024:cAMP signaling pathway)。随机选择8个DEGs(SERPINA1、MMP7、ATP2C1、ABCC1、NMT1、NAT1、CFTR和PRX)进行实时荧光定量PCR检测验证,结果显示这8个DEGs的表达模式与高通量转录组测序的结果基本一致,表明转录组数据准确可靠。【结论】在牦牛和犏牛的附睾尾部存在76个DEGs(43个DEGs上调,33个DEGs下调),显著富集在胆汁分泌通路、ABC转运器及c AMP信号通路上,与精子获能相关的DEGs有MMP7、IGFBP2和ABCC4基因,且这3个基因在犏牛附睾尾部呈下调表达,即精子获能失败可能是导致犏牛雄性不育的主要原因。

【Objective】In order to find out the differentially expressed genes(DEGs)in the epididymal cauda of yak and cattleyak,functional genes closely related to sperm maturation and storage were excavated,which laid a theoretical foundation for exploring the molecular mechanism of spermatogenesis and maturation process in cattleyak.【Method】Ta-king the epididymal cauda of yaks and cattleyaks as the research subject,high-throughput transcriptome sequencing was completed by Illumina 127-HiSeq 2000 platform,and after filtration,quality control and splicing assembly,DEGs were screened out by EBSeq according to the screening criteria of FDR<0.05 and|log_(2) Fold Change|>1,and then GO function annotation analysis and KEGG signal pathway enrichment analysis were performed.The accuracy of high-throughput tran-scriptome sequencing data was verified by real-time fluorescence quantitative PCR.【Result】A total of 76 DEGs were screened in the caput of the epididymis of yaks and calves,of which 43 DEGs were up-regulated and 33 DEGs were down-regulated.76 DEGs had annotation information in COG,GO,KEGG,KOG,NR,PFAM,Swiss-Prot and egg-NOG databases,especially the highest annotation rate in the COG and Pfam databases(88.16%).The results of GO func-tion annotation analysis showed that DEGs were annotated into three functional categories:biological process,cellular component and molecular function.KEGG signaling pathway enrichment analysis found that 76 DEGs were mainly signifi-cantly enriched in 3 signaling pathways,namely bile secretion pathway(ko04976:Bile secretion),ABC transporter(ko02010:ABC transporters)and cAMP signaling pathway(ko04024:cAMP signaling pathway).Eight DEGs(SERPI-NA1,MMP7,ATP2C1,ABCC1,NMT1,NAT1,CFTR,PRX)were randomly selected for real-time fluorescence quanti-tative PCR,and the expression patterns of the eight DEGs were basically consistent with the results of high-throughput transcriptome sequencing,indicating that the transcriptome data were accurate and reliable.【Conclusion】There are 76 DEGs(43 DEGs up-regulated and 33 DEGs down-regulated)in the epididymal cauda of yak and cattleyak,significantly enrich in bile secretion pathway,ABC transporter and cAMP signaling pathway,among which the DEGs associated with sperm capacitation include MMP7,IGFBP2 and ABCC4 genes,the expression of these genes in the epididymal cauda of cattleyak is down-regulated,that is,failure of sperm capacitation may be the main cause of male infertility in cattleyak.